Lipopolysaccharide-induced MCP-1 gene expression in rat tubular epithelial cells is nuclear factor-κB dependent
Lipopolysaccharide-induced MCP-1 gene expression in rat tubular epithelial cells is nuclear factor-κB dependent
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DOI：
10.1046/j.1523-1755.2000.00051.x
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发表时间：
2000-05-01
影响因子：
19.6
通讯作者：
Harris, DCH
中科院分区：
文献类型：
--
作者：
Wang, YP;Rangan, GK;Harris, DCH
背景。内毒素是与感染相关的急性肾衰竭发展和慢性肾炎加速的重要因素。脂多糖（LPS；内毒素的主要成分）是肾细胞产生单核细胞趋化蛋白-1 (MCP-1) 的最有效触发因素之一，单核细胞趋化蛋白-1 (MCP-1) 是一种关键细胞因子，参与急性和慢性肾脏疾病中免疫细胞募集至肾间质的过程。关于肾小管上皮细胞中 MCP-1 响应 LPS 的转录调节的知识尚不完整。方法。在原代培养的大鼠近端小管细胞 (PTC) 中研究 MCP-1 的转录调控，并将其暴露于 LPS，使用核因子 kappa B (NF-kappa B) 的电迁移分析和超位移分析以及 NF-kappa B 抑制蛋白 I kappa B 的蛋白质印迹。为了证明 NF-kappa B、激活蛋白 (AP-1) 和序列特异性转录因子 (Sp1) 的作用、突变和缺失分析使用插入转染至肾小管上皮细胞系（NRK-52E）的荧光素酶报告构建体中的大鼠MCP-1 5'-侧翼区域的3.5kb片段来进行。结果。 LPS 以剂量和时间依赖性方式增加 NF-κ B，这与 MCP-1 mRNA 表达的增加类似。 I kappa B α 在 LPS 治疗后 30 分钟内下降，但在两小时后恢复到基础水平。 I kappa B beta 水平在 LPS 刺激后一小时内下降，并在整个培养期间保持较低水平。 LPS 刺激后，转染的 MCP-1 基因 5' 侧翼区域的活性增加了近三倍。位于 5' 侧翼区域增强子区域的 NF-κ B 结合位点的突变或缺失，导致 LPS 诱导的荧光素酶活性增加完全丧失。位于 MCP-1 近端启动子区域的推定 AP-1 和 Sp1 位点的突变降低了未刺激细胞中的基础荧光素酶活性，但对 LPS 刺激的荧光素酶活性没有影响。结论。这些研究证明，NF-κ B 对于 LPS 诱导的 MCP-1 转录至关重要，而 AP-1 和 Sp1 对于大鼠肾小管细胞中 MCP-1 的基础表达至关重要。 MCP-1 转录调控的物种特异性对于制定预防内毒素介导的肾损伤的治疗方法具有重要意义。
Background. Endotoxin is an important factor in the development of acute renal failure related to infection and in acceleration of chronic nephritis. Lipopolysaccharide (LPS; the major component of endotoxin) is one of the most potent triggers for renal cells to produce monocyte chemoattractant protein-1 (MCP-1), a key cytokine involved in immune cell recruitment into the renal interstitium in acute and chronic renal diseases. Knowledge about the transcriptional regulation of MCP-1 in renal tubular epithelial cells in response to LPS is incomplete.Methods. Transcriptional regulation of MCP-1 was investigated in rat proximal tubule cells (PTCs) in primary culture and was exposed to LPS using electromobility shift assay and supershift analysis for nuclear factor-kappa B (NF-kappa B) and Western blot for the NF-kappa B inhibitory protein I kappa B. To prove the role for NF-kappa B, activator protein (AP-1), and sequence-specific transcription factor (Sp1), mutation and deletion analysis was performed using a 3.5 kb fragment of rat MCP-1 5'-flanking region inserted into a luciferase reporter construct transfected into tubular epithelial cell line (NRK-52E).Results. LPS increased NF-kappa B in a dose- and time-dependent manner, which paralleled that of MCP-1 mRNA expression. I kappa B alpha decreased within 30 minutes of LPS treatment, but returned to basal levels by two hours. I kappa B beta levels were depressed within one hour and remained low throughout the culture period after LPS stimulation. The activity of the transfected 5'-flanking region of the MCP-1 gene increased nearly threefold after LPS stimulation. Mutation or deletion of NF-kappa B binding sites, located in the enhancer region of the 5'-flanking region, resulted in a total loss of LPS-induced increase in luciferase activity. Mutation of putative AP-1 and Sp1 sites, located in the proximal promoter region of MCP-1, reduced basal luciferase activity in unstimulated cells, but had no effect on LPS-stimulated luciferase activity.Conclusions. These studies prove that NF-kappa B is critical for LPS-induced MCP-1 transcription, and AP-1 and Sp1 are essential for basal expression of MCP-1 in rat tubule cells. The species-specific nature of transcriptional regulation of MCP-1 has important implications for the delineation of treatment to prevent endotoxin-mediated renal injury.